Chapter 14 Discussion Questions and Answers

14.1 What factors are important in producing a high-quality microscope image?

Answers should include discussion about colour and aberration correction, numerical aperture, objective types and correct microscope set-up.

14.2 What techniques and microscopes could be used to examine unstained samples? Give examples of different samples and the microscope and/or technique used to visualize different samples.

Answers should include information about large tissue biopsies being examined with a stereo microscope.
Cells/tissue sections could be viewed using a darkfield or phase contrast microscope. Crystalline samples can be viewed with polarisation.

14.3 Describe how you would set up a microscope for Koehler illumination.

Fully open the condenser aperture diaphragm and place a stained slide under the 10x objective. If the condenser has a swing-in top lens then this should be swung into the light path, and the condenser height adjusted so that the top lens is close to the slide. Focus the specimen and then close the field diaphragm so that it is visible in the centre of the eyepiece field. Adjust the condenser focus control so that the edges of the field diaphragm are in sharp focus. Adjust the position of the field diaphragm so that it is in the centre of the field of view, and then open it until it disappears from the field of view. Remove an eyepiece and close the condenser aperture diaphragm so that it is approximately 70% open, then replace the eyepiece and observe the sample as required.

14.4 Describe with a diagram how an epifluorescence microscope works, and what its uses are in a biomedical laboratory.

Diagram similar to figure 8.10 with description of main components.

Uses in biomedical science:
• Immunofluorescence techniques
• FISH
• Fluorescent dye staining (eg amyloid and TB)